The present invention relates to a tube for sperm washing and concentration and to a method using the tube in order to wash and concentrate sperm collected from humans which is then used to contribute to assisted reproduction technology (ART).
The reasons for male infertility are various, but about 90% is due to poor semen quality resulting from idiopathic dysfunction of spermatogenesis. The treatment methods for male infertility are roughly classified into surgical treatment, pharmacotherapy for activation of spermatogenesis and ART. The surgical treatment includes methods such as high ligature in varicocele spermophlebectasia, seminal duct anastomosis in obstructions of the seminal tract and the like which have produced good results in the urological field. However, the effectiveness of the pharmacotherapy is not high in the case of poor semen quality, and the treatment faces hard going. Therefore, ART has been put to practical use for the treatment of male infertility. ART broadly means performing fertilization, medically assisting formation of gamete, fertilization, implantation and maintaining pregnancy. Practically, this means mostly fertilization by artificial insemination from intra-uterine insemination (IUI) to in vitro fertilization embryo transfer (IVF-ET) and further to micro-fertilization.
The number of sperm in semen ejaculated into the vagina decreases as it climbs up within the female genital tracts such as the cervical canal, uterine cavity and uterine tube. Finally, about 10 sperm reach the ampulla of the uterine tube where fertilization takes place. The meaning of semen quality becoming worse means a decrease in the number of sperm reaching the place of fertilization. Therefore, study and treatment of ART have two directions. One is improvement of the method for insemination by providing the sperm as close as possible to the oocyte by by-passing the climbing of sperm up the female genital tract, in order to enable fertilization with as few sperm as possible. In IUI, the cervical canal is by-passed. In IVF-ET, the oocyte ovum is taken out from the body and fertilized in vitro. Further in intra-cytoplasma sperm injection (ICSI), one sperm runs into the cytoplasm of oocyte by perforation, namely, fertilization is also by-passed. In other methods, washing and concentration of the ejaculated semen is attempted in order to provide as many sperm as possible for the insemination.
The most basic function of the sperm is to transport chromosomes. The sperm is functionally classified into the acrosome, sperm head and midpiece-tail. The sperm head includes chromosome. The midpiece-tail is concerned with energy metabolism and sperm motility. And the acrosome is concerned with adhesion and fusion with oocyte ovum. In general, preparation of sperm provided for artificial insemination has, as an objective, selection of progressively motile sperm being in the matured normal state, which is mainly the function of the midpiece-tail.
The sperm immediately after ejaculation has only potential fertility, but obtains the possibility of insemination through physiological and morphological changes such as capacitation, acrosome reaction and the like by culture for some hours in the female genital tract or in vitro. In IVF-ET, because the concentration of the sperm required for insemination is low, it is regarded as the best method for treating oligozoospermia and asthenozoospermia. However, in the case with poor semen quality, it is clinically clear that insemination is impossible because of few motile sperm, which suggests that an understanding of the acrosomal functions, including capability for induction of acrosome reaction, in addition to concentration and motility of sperm (the function of the midpiece-tail) which were previously regarded as an indication for fecundity fertility (namely, the fertility against female) is also important. Further, because ICSI is introduced for a severe case of poor semen quality, it is important to understand in detail the function of sperm as well as estimation of chromosome. Improvement of the method for insemination results in that the number of sperm provided for the insemination is decreased. However, in vitro operations of oocyte ovum and embryo are also required. Further, in the preparation of sperm, it is needed to perform selection of sperm and physiological change of sperm in vitro, instead of in the female genital tract. Therefore, establishment of an improved method for purifying sperm corresponding to the improvement in the method for insemination is indispensable.
The sperm provided for artificial insemination differ in the conditions for preparation according to the method of insemination. Namely, in IUI, sperm concentration is firstly required. In IVF-ET, required sperm concentration is less than that in IUI, but higher techniques such as selection of motile sperm, removal of seminal plasma and bacteria, and the like are also required (the method for sperm washing and concentration). The preparation of sperm is roughly classified into two groups, namely a method by centrifugation and a method by separation caused by the sperm""s motility. In the method by centrifugation, density gradient centrifugation was previously employed by using polymerized sucrose, Ficoll, but at present modified colloidal silica gel or Percoll, is used. As the method for insemination, the single-layered xe2x80x9cPercollxe2x80x9d method for sperm concentration, the cushion method, the multi-layered xe2x80x9cPercollxe2x80x9d method, which, enables the selection of sperm, the continuous-step density gradient centrifugation method and the like are employed. And as the method by separation caused by the sperm""s motility, the swim up method is generally employed, but in the case with poor semen quality, the swim down method, which is a variation of the swim up method, is employed.
The density gradient centrifugation method using Percoll is a method comprising, in order to avoid complications in the operation of continuous-step density gradient centrifugation, layering semen directly onto 80% Percoll which is made isotonic and stirring the layers of semen and Percoll to make a continuous density gradient for centrifugation. The motile sperm is concentrated in the sediment.
Sperm loses cytoplasm during its formation and maturation. The matured sperm having motility has a higher density than bacteria and unmatured sperm having cytoplasm. The density gradient centrifugation method using Percoll is carried out by separating the matured motile sperm from seminal plasma and bacteria, based on such a theory.
However, in the prior density gradient centrifugation method using Percoll, because the supernatant is removed by pipetting, etc., after the centrifugation, there is a defect that the concentrated sperm in sediment is contaminated again by the flow of seminal plasma or bacteria attached to the wall of centrifuge tube into the sperm. There is another defect that much Percoll remains because the amount of sediment is as much as 0.1 to 0.2 ml. Also, in artificial insemination with a husband""s semen (AIH) by injecting the washed sperm into the uterine cavity, the sediment is required to be diluted again with culture medium and centrifuged at a low rate in order to remove Percoll. As a result, the sperm recovery rate is lowered (concentration of sperm is lowered), and motility of sperm is also lowered. Thus, purification of sperm has difficulties. In the ejaculated semen, fiber of undergarment to which bacteria is attached is also contained. As a result, there is also a problem that sperm is contaminated by fiber which is contained in sediment after centrifugation.
Also, Percoll on the market has a high level of endotoxin. Therefore, such Percoll can not be used for sperm washing, and sperm suspended in Percoll can not be directly added to the embryo culture system for IVF-ET.
The present invention is accomplished in view of the above mentioned background. An object of the present invention is to provide a method for sperm washing and concentration and a tube for sperm washing and concentration by which contamination by bacteria and remaining Percoll are not caused.
An object of the present invention is also to provide a method for sperm washing and concentration and a tube for sperm washing and concentration by which a high sperm recovery rate without lowered sperm motility is obtained.
Another object of the present invention is to provide a method for sperm washing and concentration and a tube for sperm washing and concentration by which endotoxin can be removed effectively.
As the result of extensive investigations in order to solve the above mentioned problems, the present inventors found that the flow of supernatant can be inhibited by providing a small diameter portion at the bottom of the centrifuge tube in order to concentrate sperm at the bottom portion having a small diameter by centrifugation and, after centrifugation, by bending and cutting of the bottom portion having the small diameter under a condition that reduced pressure is kept in the centrifuge tube. Thence, the present invention has been accomplished.
Namely, the present invention relates to a tube for sperm washing and concentration comprising a tube having an open end and a bottom portion with a small diameter, and an elastic bladder removably provided at the open end of the tube. Herein, a vulnerable or weakened part may be further provided to easily bend and cut the bottom portion having the small diameter.
The present invention also relates to a method for sperm washing and concentration comprising the steps of:
i) filling the above mentioned glass tube for sperm washing and concentration with a density gradient carrier,
ii) sucking up semen diluted with the same volume of HANKS solution or physiological saline into a syringe,
iii) gently discharging the semen of step ii) onto a removing filter; removing fiber, gelatin-like mass and urolithiasis therein by the removing filter; and layering the filtrated semen onto the density gradient carrier in the glass tube,
iv) after layering the whole volume of the semen onto the density gradient carrier, stirring both sides of the interface between the semen and the density gradient carrier to cause the interface to disappear,
v) centrifuging the glass tube of step iv),
vi) after the centrifugation, providing the elastic bladder in a compressed state at the open end of the glass tube and obtaining the sediment containing the washed and concentrated sperm and the density gradient carrier by bending and cutting the bottom portion having a small diameter of the glass tube, and
vii) removing the layer of the density gradient carrier so as to leave only the sediment.
Herein, as the density gradient carrier, modified colloidal silica (Percoll) or polymerized sucrose (Ficoll) and the like are exemplified. Preferably, the Percoll is treated to remove endotoxins and then is added to culture medium to make it isotonic and has a 90 to 98% concentration. In the present invention, xe2x80x9cPercollxe2x80x9d means colloidal silica sol with polyvinyl pyrrolidone coating.
The condition of centrifugation may be variously selected according to the desired object, and is generally 1.000xc3x97g, for 20 to 30 minutes.